Mycobacterial genus-specific DNA probe and its expressed product

ABSTRACT

Recombinant clone pMptb #48 expressing a 36k M. paratuberculosis antigen is provided that provides a more sensitive and discriminating test than those in which crude antigens have been used. A mycobacterial genus-specific DNA probe corresponding to a 1.4 kb BamH1-DNA insert in pMptb #48 is also provided. The 1.4 kb insert includes the entire DNA coding sequence for an M. paratuberculosis 36k protein. There is also provided methods for serologically testing specimens which include the use of recombinant clone pMptb #48 and/or the p36k protein and/or fragments of the p36k protein. Also provided are methods for identifying mycobacterial infections using a probe corresponding to the 1.4 kb DNA sequence shown in FIG. 4, the coding portion of the 1.4 kb DNA sequence, or fragments thereof.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to recombinant clones specific to the genus Mycobacterium and their expression products. This invention also relates to methods for diagnosing mycobacterial infections using Mycobacterium-specific recombinant clones and their expression products.

2. Related Technology

The genus Mycobacterium consists of a diverse group of acid-fast bacilli including a number of important human and animal pathogens. Until recently, the three human pathogens M. tuberculosis, M. leprae, and M. bovis have been the most studied mycobacterial species. However, in immunocompromised patients, a number of opportunistic pathogens from the genus Mycobacterium have emerged, most notably disseminated M. avium infection in AIDS patients. In addition, there has also been a marked resurgence of human tuberculosis related to the AIDS epidemic.

The definitive diagnosis of mycobacterial infections require the isolation by culture. Pathogenic mycobacteria grow very slowly (up to six weeks in culture) and some even require animals or tissues to support their growth. Furthermore, standard protocols for definite identification include complex and time consuming procedures, especially for nontuberculous mycobacteria, which can further delay identification by several weeks.

With the emergence of an increasingly large proportion of multiple drug resistant strains of M. tuberculosis, rapid isolation and identification methods are essential for proper and efficient delivery of medical care. In a clinical laboratory setting, it would be very helpful if suspected mycobacteria-containing specimens could be evaluated rapidly and accurately. Elimination of those specimens that were negative from further laboratory analysis would reduce the labor required to process these specimens and, most importantly, free these patients from unnecessary antimycobacterial therapy until the results of their culture had been obtained.

Pathogenic mycobacteria contain a complex mixture of antigens with varied antigenic determinants and there is much cross-reactivity among mycobacteria antigens and antigens from other organisms. Many of these determinants are also expressed by heterologous mycobacterial species and as a result, antibodies to one mycobacteria species often react with others, limiting the value of diagnostic tests based on detection of crude and/or native antigens. Thus, serological diagnosis of most mycobacterial infections may only be demonstrable at the epitope level.

BRIEF DESCRIPTION OF THE INVENTION

It is an object of this invention to provide recombinant clones expressing M. paratuberculosis antigens that provide a more sensitive and discriminating test than those in which crude antigens have been used. It is another object of the present invention to provide a mycobacterial genus-specific DNA probe. Accordingly, there is provided according to the invention recombinant clone pMptb #48. Recombinant clone pMptb #48 contains a 1.4 kb BamH1-DNA insert which includes the entire DNA coding sequence for an M. paratuberculosis 36k protein. Accordingly, the present invention is also considered to include the 1.4 kb BamH1-DNA insert and the 36k protein for which it codes. The present invention is also considered to include fragments of both the 1.4 kb BamH1-DNA insert and the 36k protein. For the purposes of the present invention, and with respect to the DNA sequence, the term "fragments" shall mean oligonucleotides of at least 15 nucleotides in length which code for biologically active sites on the protein or which can be used as PCR primers. With respect to the 36k protein, the term "fragments" for the purposes of the present invention shall mean peptides of at least 8 amino acids in length which contain antigenic epitopes reactive with antibodies to the whole protein.

The present invention is also considered to include methods for serologically testing specimens which include the use of recombinant clone pMptb #48 and/or the p36k protein and/or fragments of the p36k protein. The present invention is also considered to include methods for identifying mycobacterial infections using a probe corresponding to the 1.4 kb DNA sequence shown in FIG. 4, the coding portion of the 1.4 kb DNA sequence, or fragments thereof.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of the sequencing strategy of the 1402 base pair M. paratuberculosis fragment containing p36 encoding-gene by nested deletion technique.

FIG. 2 is a summary of humoral immune response against Mycobacterium paratuberculosis p-36k recombinant clone on SDS-immunoblots.

FIG. 3 shows the bacterial strains analyzed to establish the specificity of the mycobacterial genus-probe.

FIG. 4 shows the DNA sequence of the sense strand of the 1.4 kb BamH1-DNA insert.

FIG. 5 is a linear map of the region that contains the p36K encoding gene together with the putative amino acid sequence for which it codes.

DETAILED DESCRIPTION OF THE INVENTION

We isolated, sequenced and characterized a recombinant clone pMptb #48, expressing a 36K antigen that was confirmed by in vitro transcription-translation technique combined with immunoblotting against rabbit anti-M. paratuberculosis hyperimmune serum and the nucleotide sequence of the 1402 bp that contains the p36K-encoding gene. Analyzing the recombinant clone pMptb #48 by SDS-immunoblotting technique with sera obtained from patients with mycobacterial and other diseases as well as controls, we were able serologically to group Crohn's disease (which has been linked with M. paratuberculosis) and sarcoidosis (a chronic granulomatous disease that has also be linked with mycobacterial infection) with other mycobacterial diseases. The results were: (Positive/total tested): 22/27 Crohn's disease (18.5%, 95% C.I.=62% to 94%), 4/6 tuberculosis (67%, 95% C.I.=22% to 96%), 6/6 leprosy, 5/5 sarcoidosis compared to 9/17 ulcerative colitis and 4/15 non-inflammatory bowel disease (non-IBD).

Rabbit hyperimmune anti-M. paratuberculosis serum was produced and adsorbed according to known methods (2,3). Rabbit hyperimmune anti-M. avium serum and serum from clinical cows with Johne's disease were obtained from Dr. M. Collins, University of Wisconsin, School of Veterinary Medicine, Madison, Wis. Serum from clinical sheep and goat with paratuberculosis were obtained from Allied Monitor, Inc., Fayette, Mo., The efficiency of adsorption of each serum was evaluated by immunoblotting against M. paratuberculosis sonicate and non-recombinant E. coli lysate proteins fractionated on 12% SDS-PAGE (2). As a result of antibody screening, of our M. paratuberculosis (strain Linda) expression library, a total of 24 recombinant clones that reacted with the hyperimmune rabbit-antiserum were purified and analyzed by SDS-PAGE immunoblotting technique as described previously (3). Filter strips were incubated with adsorbed serum (diluted 1:500 for rabbit antiserum, 1:30 for animal sera and 1:50 for patient sera). The color detection was visualized with appropriate Anti-lgG peroxidase conjugates and 4-chloro-1-naphthol substrate.

To investigate whether the humoral immune response of CD patients would recognize any of the recombinant clones that were picked up and purified with rabbit anti-M. paratuberculosis, copies of SDS-immunoblot strips representing all 24 individually purified recombinants were tested. Seven of these clones reacted with the pooled CD patient sera; three were known to express the 65K heat shock protein (3). From the remaining four recombinants, one expressed 36K protein (P-36K) that reacted to the CD patient sera individually.

As discussed above, to compare the humoral reactivity of CD patients and patients with other mycobacterial diseases and controls with the clone expressing the P-36K, immunoblot strips containing this recombinant protein were tested with each individual adsorbed patient's serum. The results are shown in FIG. 2. Over 75% of tuberculosis and leprosy patients, and all BCG immunized individuals gave positive reactions with the P-36K protein. When the results of CD, sarcoidosis, ulcerative colitis, non-inflammatory bowel disease controls, and BCG negative individuals were compared, CD and sarcoidosis grouped with other mycobacterial diseases in regard to reactivity to P-36K. These results mimic the results with IS900-specific PCR-assays used to detect M. paratuberculosis in resected tissues of CD patients (1,4). In addition, all sera tested from patients with sarcoidosis were also positive with P-36K.

To determine if serum from animals with Johne's disease would recognize this P-36K recombinant clone, pooled serum from 3 cows, 2 sheep, and 2 goats, all with clinical symptoms, were tested separately. Only the goat serum reacted with P-36K protein. This indicates that the humoral immune response of different species respond differently to this antigen.

The foregoing results demonstrate that the mycobacterial recombinant protein from clone pMptb #48 and antigenic fragments thereof can be used in mycobacterial serological tests and for vaccine development.

In hybridization studies, the mycobacterial 1.4 kb BamH1-DNA insert from clone pMptb #48 hybridized to all mycobacterial species and none of the 20 related and unrelated organisms tested (FIG. 3). The nucleotide sequence for the 1.4 kb insert is shown in FIG. 4. These results demonstrate that this DNA fragment and sub-fragments thereof can be used as a genus-specific probe as well as to develop genus specific polymerase chain reaction (PCR) assay for the identification of mycobacteria. The sequence of this mycobacterial DNA fragment can be used as genus-specific probe and for the development of a mycobacteria-genus specific PCR assay.

REFERENCES

4. Dell'Isola, B., C. Poyart, O. Goulet, J. Mougenot, E. Sadoun-Journo, N. Brousse, J. Schmitz, C. Ricour, P. Berche. 1994. Detection of Mycobacterium paratuberculosis by polymerase chain reaction in children with Crohn's disease. J. Infect. Dis. 169:449-451.

6. El-Zaatari, F. A. KI., L. Engstrand, D. C. Markesich, and D. Y. Graham. 1992. Screening of a Mycobacterium paratuberculosis expression library with polyclonal antiserum and amplified DNA probes to identify species-specific immunodominant antigens. In R. J. Chiodini, Kreeger J. M. (eds): Proceedings of the Third International Colloquium on Paratuberculosis. Providence, R.I., USA: International Association for Paratuberculosis Inc, pp254-268.

7. El-Zaatari, F. A. K., S. Naser, L. Engstrand, C. Y. Hachem, and D. Y. Graham. 1994. Identification and characterization of Mycobacterium paratuberculosis recombinant clones expressed in E. coli. Current Microbiology 29: (in press).

16. Sanderson, J. D., M. T. Moss, M. L. Tizard, and J. Hermon-Taylor. 1992. Mycobacterium paratuberculosis DNA in Crohn's disease tissue. Gut 33:890-896.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 2                                                   (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1402 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        GATCCCGACACCACGTGGCCGTCCGCGGAGGTCACCCGGTAGTTGGCCGTGTAGGTCCCG60                 GCCGGCCCCAGCGGGCGCACCGCCACGCTGACCACCGCGTCCTGCACGGTGGGCTCTCCG120                GTGGACCACACGTTGCCGTCCGGCCCGACCACCGTAATCGCGGCGAAGGTGGTTTGCAGC180                TGTTCGTTGAAGGTCGCGCTGACCCGGTGCGGCCCGGTCGTNAGCGCCGCTTCGGCGGCG240                GTNTCGGTCGCCACCCGGNCGGCGTGCGCCCACGACGCGGGCGCGGTCAGCCCGGCGATC300                AGGACCAGGGCGCCGACGCACGCGCCAACCGCCCAGCGCCTCATGCCCGCCGGCGGACCA360                GGGCGATCGCGATGCCCAGCGCGGCCAGCACCAGCGCGGCGCCGCCCAGCCACCGCGCGG420                CGTTGTCGGCCGCGGCGGGTCCGCCGGGGTGCTGGTGGCCGGCCGCCGGCCGGCGGCAGG480                GCGAGCGTCGGGGCCGGGTGCTCGGGCTCGCCGCCGTCGGGCAGCGGTGGCTGATCCCAC540                TTGACCACGGTGCCGTCGGCGTAGGTCTGGNTGGCCGGAAAGCTGACGGTGTCGGCGTCG600                GGCAGCTTCACCGACAGCCGGAACAGGGCGAACTGGTCGGGCCCGATGCCGCCGCCCGCG660                GGGGTTCCCAGGTGACCGAGCGGACGGTGCCGGATGCCGCGTCCCGGTCCAGCCGGGCCG720                TCCAGCCCGGCATGGTCTCGGTGTGCGCCGCGGCCACGTTGGGCAGCGCGACGGTCAGCG780                CGGTGGTGGCGGCCCCGGTGTTCGATTCGTTGGGCACCTGGAAGGTGACGACGGCCATGG840                CGCCGCGCACCGGGTTGTCGCTGCCGGCGTGCACGTGCGCCGACGCCATCGGGGACCAGG900                CCACCGATCCCAGGCACAGGGCGGCGACGGCGACCAGCAGGCGCGGGGCCCGGCGCTTGC960                GCGGCGGCATGCGGGTAGCTCAACCCGAGGCGGGCCATCAGGTCCGCCTCGATGCCGTCG1020               AGCTGCGAGGAGATGGCGGCGTGTCCCGCCCGGCGCCGGGCGGCGGGCATGTTCTCCGCG1080               GCGGTCACCGCGGCCGCCAGGTCGGTGAGCCGCTCGGAGATGGCGGCGACGAACTGCTTG1140               GTCTCGCCGTCCTTGGTTTGCGTTCCTGCACCAGCCGCAACGACTTCTCCGCCCCGGCGA1200               TGCGGGCCGACAGCTGGGCGCCGTGGCCGGAGAACTGGCCGATCTGGGCCAACGGGACGC1260               CCAGCCGATCGGCGCGGCGCTGGTCGATCAGCGCGCGCGCCGAGACGGGGCGCGGTAGAC1320               GAACGGGGTGAGGATCGGGGCGAACAGCCGGACAACGTGAGGTCCGAGCTCGGTACAAGT1380               TGGATGATAGTTGATTTCTTTG1402                                                     (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 465 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        SerArgHisHisValAlaValArgGlyGlyHisProValValGlyArg                               151015                                                                         ValGlyProGlyArgProGlnArgAlaHisArgHisAlaAspHisArg                               202530                                                                         ValLeuHisGlyGlyLeuSerGlyGlyProHisValAlaValArgPro                               354045                                                                         AspHisArgAsnArgGlyGluGlyGlyLeuGlnLeuPheValGluGly                               505560                                                                         ArgAlaAspProValArgProGlyArgXaaArgArgPheGlyGlyGly                               65707580                                                                       XaaGlyArgHisProXaaGlyValArgProArgArgGlyArgGlyGln                               859095                                                                         ProGlyAspGlnAspGlnGlyAlaAspAlaArgAlaAsnArgProAla                               100105110                                                                      ProHisAlaArgArgArgThrArgAlaIleAlaMetProSerAlaAla                               115120125                                                                      SerThrSerAlaAlaProProSerHisArgAlaAlaLeuSerAlaAla                               130135140                                                                      AlaGlyProProGlyCysTrpTrpProAlaAlaGlyArgArgGlnGly                               145150155160                                                                   GluArgArgGlyArgValLeuGlyLeuAlaAlaValGlyGlnArgTrp                               165170175                                                                      LeuIleProLeuAspHisGlyAlaValGlyValGlyLeuXaaGlyArg                               180185190                                                                      LysAlaAspGlyValGlyValGlyGlnLeuHisArgGlnProGluGln                               195200205                                                                      GlyGluLeuValGlyProAspAlaAlaAlaArgGlyGlySerGlnVal                               210215220                                                                      ThrGluArgThrValProAspAlaAlaSerArgSerSerArgAlaVal                               225230235240                                                                   GlnProGlyMetValSerValCysAlaAlaAlaThrLeuGlySerAla                               245250255                                                                      ThrValSerAlaValAlaAlaProValPheAspSerLeuGlyThrTrp                               260265270                                                                      LysValThrThrAlaMetAlaProArgThrGlyLeuSerLeuProAla                               275280285                                                                      CysThrCysAlaAspAlaIleGlyAspGlnAlaThrAspProArgHis                               290295300                                                                      ArgAlaAlaThrAlaThrSerArgArgGlyAlaArgArgLeuArgGly                               305310315320                                                                   GlyMetArgValAlaGlnProGluAlaGlyHisGlnValArgLeuAsp                               325330335                                                                      AlaValGluLeuArgGlyAspGlyGlyValSerArgProAlaProGly                               340345350                                                                      GlyGlyHisValLeuArgGlyGlyHisArgGlyArgGlnValGlyGlu                               355360365                                                                      ProLeuGlyAspGlyGlyAspGluLeuLeuGlyLeuAlaValLeuGly                               370375380                                                                      LeuArgSerCysThrSerArgAsnAspPheSerAlaProAlaMetArg                               385390395400                                                                   AlaAspSerTrpAlaProTrpProGluAsnTrpProIleTrpAlaAsn                               405410415                                                                      GlyThrProSerArgSerAlaArgArgTrpSerIleSerAlaArgAla                               420425430                                                                      GluThrGlyArgGlyArgArgThrGlyXaaGlySerGlyArgThrAla                               435440445                                                                      GlyGlnArgGluValArgAlaArgTyrLysLeuAspAspSerXaaPhe                               450455460                                                                      Leu                                                                            465                                                                            __________________________________________________________________________ 

I claim:
 1. Recombinant clone pMptb #48.
 2. An isolated nucleic acid comprising nucleotides in a sequence selected from the group consisting of the sequence of nucleotides 1-1402 of SEQ ID NO. 1 and the sequences of fragments thereof at least 15 nucleotides in length.
 3. The isolated nucleic acid of claim 2, wherein said nucleotides are in a sequence selected from the group consisting of the sequence of nucleotides 372-1328 of SEQ ID NO. 1 and the sequences of fragments thereof at least 15 nucleotides in length.
 4. A method for assessing the presence in a sample of nucleotide sequences which bind with a mycobacterial genus-specific nucleic acid probe comprising:contacting a nucleic acid extract from said sample with a nucleic acid probe comprising nucleotides in a sequence selected from the group consisting of the sequence of nucleotides 1-1402 of SEQ ID NO. 1 and the sequences of fragments thereof at least 15 nucleotides in length; allowing sufficient time for hybridization to occur between said nucleic acid probe and any complementary sequences in said sample of nucleotide sequences; and detecting the presence of any hybridization between said nucleic acid probe and complementary sequences in said sample of nucleotide sequences.
 5. A method for amplifying DNA which codes for the protein coded by nucleotides 372-1328 of SEQ ID NO. 1, comprising:selecting oligonucleotide primers designed on the basis of the sequence of nucleotides 1-1402 of SEQ ID NO. 1 or fragments thereof; and using polymerase chain reaction with said oligonucleotide primers to amplify said DNA which codes for the protein coded by nucleotides 372-1328 of SEQ ID NO.
 1. 